Through forward genetics screens in haploid human cells harboring a fluorescent reporter of WNT signaling, we discovered regulators of the intact pathway as well as those selectively required to sustain oncogenic signaling. This study (Lebensohn et al., eLife 2016) comprised seven genome-wide screens systematically interrogating the pathway, including screens for positive, negative, and attenuating regulators of ligand-induced signaling, as well as suppressor screens following disruption of key regulators commonly mutated in WNT-driven tumors, such as the tumor suppressor APC. A comparative analysis of the screens revealed new regulatory mechanisms for ligand reception, signal transduction and transcriptional activation. Our current goal is to elucidate the molecular underpinnings of these new regulatory mechanisms, understand their physiological functions and evaluate their potential as therapeutic targets. Since the inception of my laboratory in October of 2018, our specific objective has been to understand a new mechanism of WNT signal transduction independent of changes in beta-catenin levels. Beta-catenin is the main transcriptional co-activator in the WNT pathway, and regulation of beta-catenin abundance by a multi-protein assembly called the destruction complex is considered the principal control point in the WNT signaling cascade. Binding of WNT ligands to their co-receptors triggers inactivation of the destruction complex, which results in accumulation of beta-catenin and expression of WNT target genes. Mutations in components of the destruction complex that lead to aberrant accumulation of beta-catenin are a major driver of tumorigenesis in many types of cancer. Identifying mechanisms that can revert constitutive WNT signaling in cells with compromised destruction complex function is therefore of great therapeutic interest. Genetic suppressor screens are a powerful way to reveal such mechanisms, as they can illuminate intricate connections in signaling networks. We previously reported a comprehensive, comparative forward genetic analysis of WNT signaling using a haploid human cell line containing a fluorescent reporter of WNT activity (Lebensohn et al., eLife 2016). In a suppressor screen to identify genes required for constitutive WNT signaling induced by loss of the destruction complex component casein kinase 1alpha (CK1alpha), we identified HUWE1. HUWE1 encodes a HECT domain-containing ubiquitin ligase with diverse cellular functions, including regulation of cell proliferation and differentiation, apoptosis, DNA repair, and stress responses. We found that eliminating HUWE1 in cells lacking CK1alpha leads to a 90% reduction of WNT reporter activity and to an 80-85% reduction in expression of the endogenous WNT target gene AXIN2, but surprisingly, only to a 20-32% reduction of beta-catenin protein abundance (Lebensohn et al., eLife 2016). These results suggest that HUWE1 regulates WNT signaling through an additional mechanism distinct from the control of beta-catenin protein degradation. Consistent with this hypothesis, eliminating HUWE1 does not reduce WNT signaling activity induced by loss of APC or by inhibition of GSK3, two other components of the beta-catenin destruction complex. Furthermore, eliminating HUWE1 in wild type cells does not affect target gene expression or beta-catenin accumulation induced by addition of WNT ligands. These results suggest that the signaling activity caused by loss of CK1alpha is not equivalent to the signaling activity caused by loss or inhibition of other components of the destruction complex, or by WNT ligands. The former is largely dependent on HUWE1, while the latter is not. Based on these results, we propose that CK1alpha plays a unique role in negatively regulating WNT signaling in addition to its role in promoting beta-catenin degradation as part of the destruction complex. We are testing this hypothesis through biochemical, genetic and cell biological analyses. We designed and executed new forward genetic screens in haploid human cells to identify additional components of the WNT pathway responsible for mediating the effects of HUWE1 on WNT signaling in the absence of CK1alpha. In a control screen, which yielded many expected hits, we demonstrated that our current approach is valid. In the experimental screen we identified several new hits with very high statistical significance. We are currently validating and characterizing these hits.